The present invention concerns methods for synthesis and accumulation of fructose polymers in transgenic maize (Zea mays L.) by selective expression of plant-derived fructosyltransferase genes.
Higher plants accumulate various commercially useful carbohydrate polymers such as cellulose, starch and fructan. Starch and cellulose are currently used in numerous food and non-food applications in their native form, but are more likely to be enzymatically or chemically modified, which greatly expands their usefulness.
Fructans are linear or branched polymers of repeating fructose residues. The number of residues contained in an individual polymer, also known as the degree of polymerization (DP), varies greatly depending on the source from which it is isolated. For example, fructan synthesized by fungal species, such as in Aspergillus syndowi may contain only two or three fructose residues. By contrast, polymers with a DP of 5000 or greater are synthesized by several bacterial lines, including Bacillus amyloliquefaciens and Streptococcus mutans. Intermediate sized fructan, with a DP of 3 to 60, are found in over 40,000 plant species (Science and Technology of Fructans, (1993) M. Suzuki and N. Chatterton, eds. CRC Press Inc., Boca Raton, Fla., pp. 169-190).
Regardless of size, fructose polymers are not metabolized by humans. Because of this, and due to their relative sweetness, small fructans with a DP of 3-4 are used in a wide variety of low calorie food products. Polymer size is critical to its commercial use. High DP polymers are not sweet, however, they do provide texture to food products very similar to that of fat. High DP fructan used as a fat replacer also contributes very little to the caloric value of the product.
Fructans are also considered to be an excellent source of fructose for the production of high fructose syrup (Fuchs, A. (1993) in Science and Technology of Fructans, M. Suzuki and N. Chatterton, eds. CRC Press Inc., Boca Raton, Fla., pp. 319-352). Simple hydrolysis of fructan into individual fructose residues has a tremendous advantage over the current, technically demanding process of enzymatically converting starch into high fructose syrup. Using fructan as the starting material would, therefore, significantly reduce production costs.
The commercial potential for fructan is extremely high, however, its use is severely limited due mainly to the high cost of production. Fructan used in low-calorie foods is currently produced by fermentation culture. Larger polymers synthesized by bacteria are not currently produced on a commercial scale. Isolation from plants would reduce the production costs, but fructan is not found in many crops of agricultural importance. Traditional crops, adapted to wide growing regions, such as oat, wheat and barley accumulate fructan, but only at extremely low levels. Fructan is currently harvested from plants on a relatively small commercial scale and only from a single plant species, Cichorium intybus. 
Transgenic crops accumulating fructan through expression of chimeric fructosyltransferase (FTF) genes would have a significant advantage over native fructan-storing plants by making use of established breeding programs, pest resistance and adaptation to a variety of growing regions throughout the world. Examples of fructan synthesis in transgenic plants containing genes from bacterial species, such as Bacillus, Streptococcus and Erwinia have been reported (Caimi et al., (1996) Plant Physiol. 110:355-363; Ebskamp et al., (1994) Biotechnol. 12:272-275; Rober et al., (1996) Planta 199:528-536). Synthesis of fructan in these non-fructan-storing plants was demonstrated, but accumulation was often very low and in tissues where high levels of fructan were reported to have a detrimental effect on plant development.
Several important differences between transgenic plants expressing chimeric bacterial FTF genes and native fructan-storing plants were reported. The most obvious difference was in the size of the polymers synthesized. Transgenic lines containing bacterial FTF genes accumulate fructan with a DP of greater than 5000 (Ebskamp et al., (1994) Biotechnol. 12:272-275; Caimi et al., (1996) Plant Physiol. 110:355-363). Polymers synthesized in transgenic plants are, therefore, several thousand times larger than fructans which accumulate in plants such as chicory (Cichorium intibus L.) and Jerusalem artichoke (Helinathus tuberosus L.).
Differences in the specificity for donor and acceptor molecules have also been reported for bacterial and plant FTFs. The bacterial enzymes are known to release significant amounts of fructose to water as an acceptor (invertase activity), whereas the plant enzymes do not have invertase activity (Chambert, R. and Petit-Glatron, M. (1993) in Inulin and Inulin Containing Crops, A. Fuchs ed. Elsevier Press, Amsterdam. pp. 259-266). Fructose, liberated from sucrose by invertase activity, can not be used to increase the length of a polymer. Bacterial FTFs, therefore, convert sucrose to fructan less efficiently than do the plant enzymes.
The two classes of FTFs also differ in their affinity for sucrose, the sole substrate. Jerusalem artichoke sucrose-sucrose-fructosyltransferase (SST) has a Km for sucrose reported to be approximately 100 mM (Koops, A. and Jonker, H., (1994) J. Exp. Bot. 45:1623-1631). By contrast, the bacterial enzyme has a much lower Km of approximately 20 mM (Chambert, R., and Petit-Glatron, M. (1991) Biochem. J. 279:35-41). This difference may have a critical effect on fructan synthesis, resulting in higher or lower levels of accumulation, depending on the concentration of sucrose in the cell. The fundamental differences between FTF enzymes prevents meaningful predictions regarding the outcome of expression of plant genes in transgenic tissue, based on expression of bacterial FTF genes.
Predicting whether or not fructan would accumulate in a transgenic line containing the plant-derived FTF genes could be significantly enhanced if a greater understanding of the fructan metabolic pathway in native fructan-storing plants existed. The currently accepted model for fructan synthesis in plants suggests that synthesis is a two step reaction. The initial reaction involves the enzyme sucrose-sucrose-fructosyltransferase (SST). SST catalyzes the synthesis of a trisaccharide from two sucrose residues. The second step, chain elongation, is carried out by the enzyme fructan-fructan-fructosyltransferase (FFT), (Edelman J., and Jefford T. (1968) New Phytol. 67:517-531. The model has been applied to all fructan-storing plants (ca 45000 species). However, it is based largely on data from a single species, Helianthus tuberosus, and has undergone several revisions. A recent study demonstrates that the SST can act alone in producing long chain fructan (Van der Ende, W. and Van Laere, A., (1996) J. Exp. Bot. 47:1797-1803). Thus, additional revisions in the model are necessary and suggests that there is only a rudimentary knowledge of fructan synthesis in plants.
Examples of fructan synthesis in transgenic plants containing microbial or plant-derived FTF genes has been reported (Vijn, et al., (1997) The Plant J. 11:387-398; Smeekens et al., WO 96/01904; Van Tunen et al., WO 96/21023; Sevenier et al., (1998) Nature Biotechnology 16:843-846). This previous work involves expression of microbial or plant-derived SST genes only in transgenic dicotyledenous (dicots) plants. The present invention describes a method of increasing the level of fructan synthesis in transgenic monocotyledonous plants containing plant-derived SST genes or plant-derived SST and FFT genes.
Numerous differences between monocotyledonous (moncots) plants and dicots exist which inhibit useful extrapolation of events occurring in one plant based on data from another. These differences include, but are not limited to, the competition for sucrose as an energy source among biosynthetic pathways in various plant organs and among biosynthetic pathways in different plant species.
Dicots and monocots are known to differ significantly in the transport and metabolism of carbohydrate. For example, pea (Pisum sativum L.), a dicot, transports glucose-6-phosphate into amyloplasts, the site where starch synthesized and stored. In monocots, such as maize, ADPglucose is transported into the amyloplast (Denyer et al., (1996) Plant Phys. 112:779-785). This seemingly simple difference illustrates a profound difference in the metabolic pathways necessary for processing the various forms of carbohydrate transported into the amyloplast in the two separate plants.
Transport of sucrose in plants also differs among plant species. Specialized cells (basal endosperm transfer cells or BET cells) are adapted for the transport and metabolism of sucrose in maize kernels. The majority (greater than 90%) of sucrose transported to maize seeds is believed to be hydrolyzed in the specialized BET layer (Shannon, J. (1972) Plant Physiol. 49:198-202). The resulting hexose sugars are transported to the developing endosperm cells and resynthesized as sucrose prior to entering the starch biosynthetic pathway. In contrast to maize, sucrose is directly transported to tubers of potato plants and enters the starch pathway unhydrolyzed (Oparka, K. and Wright, K. (1988) Planta 174:123-126).
Although poorly understood, exploiting the differences between monocots and dicots could not be considered a new concept. These differences are what drives the commercialization of herbicides such as 2,4-D which is tremendously toxic to dicots, but has no effect on monocot species. In this light, it seems clear that recent examples of transgenic dicot species containing a plant derived FTF gene (Vijn, et al., (1997) The Plant J. 11:387-398; Smeekens et al., WO 96/01904; Van Tunen et al., WO 96/21023; Sevenier et al., (1998) Nature Biotechnology 16:843-846) can have no bearing on predicting the successful expression of FTF genes in moncot species. Variations in carbohydrate concentration, transport and metabolism among plant species, especially between moncots and dicots, are clearly too great to allow generalization.
This invention discloses a method for producing fructose polymers of various lengths through expression of plant-derived FTF genes in a transgenic monocot species. More specifically, the invention describes a chimeric gene comprising a tissue specific promoter, operably linked to the coding sequence for a sucrose-sucrose-fructosyltransferase gene (SST; EC 2.4.1.99) such that said chimeric gene is capable of transforming a monocot plant cell resulting in production of fructan with no deleterious effect on the said plant cell.
The invention further describes a chimeric gene comprising a tissue specific promoter, operably linked to the coding sequence for a fructan-fructan-fructosyltransferase gene (FFT; EC 2.4.1.100) such that said chimeric gene is capable of transforming a transformed plant cell (harboring a chimeric gene comprising a tissue specific promoter, operably linked to the coding sequence for a sucrose-sucrose-fructosyltransferase gene (SST; EC 2.4.1.99)) resulting in production of fructan, with no deleterious effect on the said plant cell.
The invention also includes a monocot plant transformed with one or both of the chimeric genes described above, such that the plant produces fructan. The invention also concerns a method of producing fructose or fructose polymers comprising growing the plant, harvesting the plant, and extracting fructan from the harvested plant.
The invention further describes a chimeric gene comprising a tissue specific promoter, operably linked to the coding sequence for a sucrose-sucrose-fructosyltransferase gene (SST; EC 2.4.1.99) such that the chimeric gene is capable of transforming a monocot plant cell resulting in production of fructose polymers containing 2 to 3 fructose residues, with no deleterious effect on the transformed plant cell.
The present invention is not limited to naturally occurring fructosyl-transferases but may equally well be performed by using modified versions thereof. Modifications may influence the activity of the fructosyltransferase in such a way that, for example, the degree of polymerization or the structure of the fructan produced is altered. Furthermore, according to the present invention a single fructosyltransferase gene or a combination of fructosyltransferase genes of plant origin may be used.
The induced accumulation of fructans in transgenic plants using the principles described herein will allow for the extraction of fructans from these plants for the purpose of fructan production. Fructans can accumulate in these plants (e.g., in harvestable organs such as roots, leaves, stems and seeds). Furthermore, the present invention further relates to seeds, cuttings or other parts of the transgenic plants which are useful for the continuous production of further generations of said plants.
The fructans produced using transgenic plants of the present invention may be used in various food and non-food applications. Examples include but are not limited to human and animal food products, in the production of fructose syrups, in the production of chemicals and plastics either as such or in a modified form.
Genetically modified crop plants which incorporate the fructosyl-transferase-encoding constructs mentioned above will allow for the efficient production of high quality carbohydrate polymers useful to man.